This invention relates to tumor metastasis-inhibiting monoclonal antibodies as well as antigens that are recognized by such antibodies. Compositions and methods are provided that are useful for treating and diagnosing metastatic tumors.
Malignant tumors shed cells which migrate to new tissues and create secondary tumors; a benign tumor does not generate secondary tumors. The process of generating secondary tumors is called metastasis and is a complex process in which tumor cells colonize sites distant from the primary tumor. Tumor metastasis remains the major cause of deaths in cancer patients, yet the molecular mechanisms underlying tumor cell dissemination are not clearly understood. There is a need to identify new molecules that are functionally involved in cancer cell dissemination in order to gain insights into the basics of metastatic spread, and to identify novel prognostic indicators and/or therapeutic targets.
Metastasis is a multi-step process in which tumor cells must detach from the primary tumor, invade the cellular matrix, penetrate through blood vessels, thus enter the circulatory system (intravasate), arrest at a distant site, exit the blood stream (extravasate), and grow. See, e.g., G. L. Nicolson (1982) Biochim. Biophis. Acta. 695: 113-176; G. L. Nicolson and G. Poste (1983) In. Rev. Exp. Pathol. 25: 77-181; G. Poste and I. J. Fidler (1980) Nature 283: 139-145; and E. Roos (1984) Biochim. Biophis. Acta. 738: 263-284. Given the complexity of the process, it is likely that there are a number of genes which mediate tumor cell metastasis. Indeed, the metastatic phenotype has been correlated with expression of a variety of proteins, including proteases, adhesion molecules, and the like. However, evidence that a given protein is directly involved in dissemination is often lacking, or difficult to prove. L. A. Liotta and W. Stetler-Stevenson (1989) J. Natl. Cancer Inst. 81: 556-557.
The human epidermoid carcinoma, HEp-3, provides a unique model system with which to detect and characterize those genes which effect metastatic dissemination. HEp-3 cells, propagated by serial passage on the chick chorioallantoic membrane (CAM), are both tumorigenic and spontaneously metastatic (T+M+). L. Ossowski and E. Reich (1980a) Cancer Res. 40: 2300-2309. However, when cells are grown continuously in vitro, they readily form primary tumors, but progressively become non-metastatic (T+Mxe2x88x92) with time. L. Ossowski and E. Reich (1980b) Cancer Res. 40: 2310-2315. With prolonged cultivation in vitro, they eventually become non-tumorigenic also (Txe2x88x92Mxe2x88x92). Loss of metastatic ability is reversible. T+Mxe2x88x92 cells carried on the CAM for two to three passages regain the ability to form spontaneous metastases. Thus, by altering growth conditions, the metastatic potential of these cells can be manipulated by the investigator.
Human urokinase-type plasminogen activator (uPA) was shown to be directly involved in dissemination of HEp-3, as spontaneous metastasis of HEp-3 cells in the chick embryo was inhibited by antibodies that were specific for human uPA. L. Ossowski and E. Reich (1983b) Cell 35: 611-619. Subsequently, it was observed that inhibition of uPA activity blocked infiltration of the CAM mesenchyme by individual HEp-3 cells. L. Ossowski (1988a) Cell 52: 321-328. However, active uPA appeared to be required for tumor cell intravasation but not extravasation. L. Ossowski (1988a). Thus, some other factor(s) must be also involved in HEp-3 dissemination. J. P. Quigley et al. (1988) Ciba Foundation Symposium 141: 22-47 and Brooks et al. (1993) J. Cell Biol. 122 (6): 1351-1359 describe the generation of monoclonal antibodies (mAbs), using xe2x80x9csubtractive immunizationxe2x80x9d, which recognize cell surface antigens expressed on HEp-3 cells and inhibit tumor metastasis in the CAM model.
Angiogenesis is the growth of new blood vessels from pre-existing vessels. Endothelial cells which line the vessel walls degrade the surrounding basement membrane and migrate into the surrounding connective tissue. The migrating endothelial cells form cords, which eventually develop lumena and become patent vessels. Angiogenesis plays a vital role in normal physiological processes (e.g., would healing, ovulation). Inappropriate angiogenesis is involved in a number of pathological processes, such as cancer metastasis, diabetic retinopathy, etc.
The present invention has generated monoclonal antibodies by subtractive immunization, which inhibit tumor metastasis and/or angiogenesis. The antigens that are recognized by such monoclonal antibodies have been identified as well.
One embodiment of the present invention provides tumor metastasis-inhibiting monoclonal antibodies 41-2 and 50-6. Functional derivatives and fragments of such monoclonal antibodies are also contemplated by the present invention.
Another embodiment of the present invention provides hybridoma cell lines 41-2 (ATCC #PTA-226) and 50-6 (ATCC #PTA-227), which produce mAb 41-2 and mAb 50-6, respectively.
Another embodiment of the present invention provides the antigens recognized by the monoclonal antibodies of the present invention, more particularly, the target antigen of mAb 41-2.
Another embodiment of the present invention is directed to antibodies raised against the target antigen of mAb 41-2, as well as antibodies raised against the target antigen of mAb 50-6.
Another embodiment of the present invention is directed to nucleic acid molecules coding for the target antigen of mAb 41-2 or a portion thereof. Expression vector and host cells are also contemplated by the present invention.
In a further aspect of the present invention, pharmaceutical compositions are provided.
A pharmaceutical composition of the present invention can include the PETA-3 antigen or a portion thereof, or the antigen recognized by mAb 41-2 or a portion thereof.
A pharmaceutical composition of the present invention can also include an antibody directed against PETA-3 or the target antigen of mAb 41-2.
A preferred pharmaceutical composition of the present invention includes a tumor-metastasis-inhibiting antibody. Preferred tumor-metastasis-inhibiting antibodies include mAb 41-2, mAb 50-6 and mAb 1A-5.
A further aspect of the invention provides methods of diagnosing metastatic tumors in a subject by detecting the expression of at least one of the metastasis-associated antigens identified by the present invention, including the PETA-3 antigen or the antigen recognized by mAb 41-2. The detection can be accomplished by using an antibody or a nucleic acid probe.
Another embodiment of the present invention provides methods of treating a metastatic tumor in a subject by administering to the subject a therapeutically effective amount of a tumor metastasis-inhibiting antibody of the present invention.
Another embodiment of the present invention provides methods of treating a metastatic tumor in a subject by administering to the subject a therapeutically effective amount of an antisense molecule of the PETA-3 gene or the gene encoding the target antigen of mAb 41-2.
In still another embodiment, the present invention is directed to methods of treating aberrant PETA-3-dependent angiogenesis in a subject by administering a therapeutically effective amount of an angiogenesis-inhibiting antibody directed against PETA-3 or a functional derivative of such antibody.